Protein-RNA Interaction
Interactions between proteins and RNA molecules are of biological and clinical importance. Proteins are complex macromolecules made up of covalently linked chains of amino acids. Each protein assumes a unique three dimensional shape determined principally by its sequence of amino acids. Many proteins consist of smaller units termed domains, which are continuous stretches of amino acids able to fold independently from the rest of the protein. Some of the important forms of proteins are as enzymes, polypeptide hormones, nutrient transporters, structural components of the cell, hemoglobins, antibodies, nucleoproteins, and components of viruses.
RNA (ribonucleic acid) is the transcription product of a DNA sequence. RNA is typically classified as either ribosomal RNA (rRNA), transfer RNA (tRNA), or messenger RNA (mRNA). RNAs are generally synthesized by enzymes that copy the nucleotide sequences from a DNA template, and the vast majority participate in protein synthesis. Ribosomal RNA is found in ribosomes which are the particles on which protein synthesis takes place. Messenger RNA is an intermediary sequence that transfers genetic information from the DNA to the ribosome. Transfer RNA carries amino acids to the site of protein synthesis. Other RNAs may be present in the prokaryotic or eukaryotic cell but occur in smaller amounts and may participate in functions such as DNA synthesis and the cutting and splicing of RNA sequences.
A certain subgroup of proteins is known to bind RNA molecules. For example, Frankel, et al. (Cell 67:1041-1046, 1991) reviewed RNA-protein interactions. Protein-RNA interactions are important in a variety of biological and clinical contexts. These interactions include infections by RNA viruses, translation and mRNA splicing. Therefore, understanding these interactions and selecting inhibitors and activators is essential when seeking RNAs as pharmaceuticals and planning rational drug design.
A variety of approaches have been used to study RNA-protein interactions. In vitro approaches include physical methods, such as x-ray crystallography, and biochemical assays, such as chemical and enzymatic footprinting, gel retardation and filter binding experiments (summarized in Frankel et al., supra). In vivo approaches to assaying RNA-protein interactions in a generally applicable manner, relying merely on binding and not on any other biological property of the molecule, are few. Binding of an RNA-binding protein to an appropriately placed site, at a suitable position upstream of the translation initiation codon in a reporter gene, can cause detectable repression of a reporter gene in yeast in vivo (Stripecke, et al., Molec. and Cell. Biol. 14:5898-5909, 1994).
Transcriptional Activation through Separated Domains
There is evidence that transcription can be activated through the use of two functional domains of a transcription factor: a domain that recognizes and binds to a specific site on the DNA and a domain that is necessary for activation, as reported by Keegan, et al., Science 231:699-407 (1986) and Ma and Ptashne, Cell 48:847-853 (1987). The transcriptional activation domain is thought to function by contacting other proteins involved in transcription. The DNA-binding domain appears to function to position the transcriptional activation domain on the target gene which is to be transcribed. In several cases now known, these two functions (DNA-binding and activation) reside on separate proteins. One protein binds to the DNA, and the other protein, which activates transcription, binds to the DNA-bound protein, as reported by Tijan and Maniatis, Cell 77:5-8, 1994.
Transcriptional activation has been studied using the GAL4 protein of the yeast Saccharomyces cerevisiae. The GAL4 protein is a transcriptional activator required for the expression of genes encoding enzymes of galactose utilization, see Johnston, Microbiol. Rev. 51:458-476 (1987). It consists of an N-terminal domain which binds to specific DNA sequences designated UAS.sub.G ("UAS" stands for upstream activation site; "G" indicates the galactose genes) and a C-terminal domain containing acidic regions, which is necessary to activate transcription, see Keegan, et al. (1986), supra, and Ma and Ptashne (1987), supra. As discussed by Keegan, et al., the N-terminal domain binds to DNA in a sequence-specific manner but fails to activate transcription. The C-terminal domain cannot activate transcription because it fails to localize the UAS.sub.G, see for example, Brent and Ptashne, Cell 43:729-736 (1985). However, Ma and Ptashne have reported (Cell 51:113-119, 1987; Cell 55:443-446, 1988) that when both the GAL4 N-terminal domain and C-terminal domain are fused together in the same protein, transcriptional activity is induced.
Other proteins also function as transcriptional activators via the same mechanism. For example, the GCN4 protein of Saccharomyces cerevisiae (as reported by Hope and Struhl, Cell 46:885-894, 1986), the LEX A protein (as a LEXA-GAL4 protein reported by Brent and Ptashne, Cell 43:729-736, 1985), the VP16 protein of herpes simplex virus (as a GAL4-VP16 hybrid reported by Sadowski, et al., Nature 335:563-564, 1988), the ADR1 protein of Saccharomyces cerevisiae as reported by Thukral, et al., Molecular and Cellular Biology 9:2360-2369, 1989 and the human estrogen receptor, as discussed by Kumar, et al., Cell 51:941-951, 1987 contain separable domains for DNA binding and for maximal transcriptional activation.
U.S. Pat. No. 5,283,173 (Fields and Song, issued Feb. 1, 1994) discloses a system to detect protein-protein interactions through use of chimeric genes which express hybrid proteins. This system uses the separation of transcription factors described above in an assay system.
None of the aforementioned articles suggest such a genetic system designed to detect protein-RNA interactions in vivo using transcriptional activation as an assay.